The family is divided into evolutionarily related groups with slightly differentsubstrate preferences, broadly designated ribonuclease H1 and H2.[2] Thehuman genome encodes both H1 and H2. Human ribonuclease H2 is a heterotrimeric complex composed of three subunits, mutations in any of which are among the genetic causes of arare disease known asAicardi–Goutières syndrome.[3] A third type, closely related to H2, is found only in a fewprokaryotes,[4] whereas H1 and H2 occur in alldomains of life.[4] Additionally, RNase H1-likeretroviral ribonuclease H domains occur in multidomainreverse transcriptase proteins, which are encoded byretroviruses such asHIV and are required for viral replication.[5][6]
In eukaryotes, ribonuclease H1 is involved inDNA replication of themitochondrial genome. Both H1 and H2 are involved in genome maintenance tasks such as processing ofR-loop structures.[2][7]
RNases H can be broadly divided into two subtypes, H1 and H2, which for historical reasons are given Arabic numeral designations ineukaryotes and Roman numeral designations inprokaryotes. Thus theEscherichia coli RNase HI is a homolog of theHomo sapiens RNase H1.[2][7] InE. coli and many other prokaryotes, thernhA gene encodes HI and thernhB gene encodes HII. A third related class, called HIII, occurs in a fewbacteria andarchaea; it is closely related to prokaryotic HII enzymes.[4]
Comparison of the structures of representative ribonuclease H proteins from each subtype. In theE. coli protein (beige, top left), the four conserved active site residues are shown as spheres. In theH. sapiens proteins, the structural core common between the H1 and H2 subtypes is shown in red. Structures are rendered from:E. coli,PDB:2RN2;T. maritima,PDB:303F;B. stearothermophilus,PDB:2D0B;H. sapiens H1,PDB:2QK9;H. sapiens,PDB:3P56.
RNases H2 are larger than H1 and usually have additional helices. Thedomain organization of the enzymes varies; some prokaryotic and most eukaryotic members of the H1 group have an additional small domain at theN-terminus known as the "hybrid binding domain", which facilitates binding to RNA:DNA hybrid duplexes and sometimes confers increasedprocessivity.[2][7][11] While all members of the H1 group and the prokaryotic members of the H2 group function as monomers, eukaryotic H2 enzymes are obligateheterotrimers.[2][7] Prokaryotic HIII enzymes are members of the broader H2 group and share most structural features with H2, with the addition of an N-terminalTATA box binding domain.[7] Retroviral RNase H domains occurring in multidomainreverse transcriptase proteins have structures closely resembling the H1 group.[5]
RNases H1 have been extensively studied to explore the relationships between structure and enzymatic activity. They are also used, especially theE. coli homolog, asmodel systems to studyprotein folding.[12][13][14] Within the H1 group, a relationship has been identified between higher substrate-binding affinity and the presence of structural elements consisting of a helix and flexible loop providing a larger and morebasic substrate-binding surface. The C-helix has a scattered taxonomic distribution; it is present in theE. coli and human RNase H1 homologs and absent in the HIV RNase H domain, but examples of retroviral domains with C-helices do exist.[15][16]
Ribonuclease H enzymes cleave thephosphodiester bonds ofRNA in a double-stranded RNA:DNA hybrid, leaving a3'hydroxyl and a5'phosphate group on either end of the cut site with a two-metal-ion catalysis mechanism, in which two divalent cations, such as Mg2+ and Mn2+, directly participate in the catalytic function.[17] Depending on the differences in their amino acid sequences, these RNases H are classified into type 1 and type 2 RNases H.[7][18] Type 1 RNases H have prokaryotic and eukaryotic RNases H1 and retroviral RNase H. Type 2 RNases H have prokaryotic and eukaryotic RNases H2 and bacterial RNase H3. These RNases H exist in a monomeric form, except for eukaryotic RNases H2, which exist in a heterotrimeric form.[19][20] RNase H1 and H2 have distinctsubstrate preferences and distinct but overlapping functions in the cell. In prokaryotes and lower eukaryotes, neither enzyme isessential, whereas both are believed to be essential in higher eukaryotes.[2] The combined activity of both H1 and H2 enzymes is associated with maintenance ofgenome stability due to the enzymes' degradation of the RNA component ofR-loops.[21][22]
Ribonuclease H1 enzymes require at least fourribonucleotide-containingbase pairs in a substrate and cannot remove a single ribonucleotide from a strand that is otherwise composed of deoxyribonucleotides. For this reason, it is considered unlikely that RNase H1 enzymes are involved in the processing ofRNA primers fromOkazaki fragments duringDNA replication.[2] RNase H1 is not essential in unicellular organisms where it has been investigated; inE. coli, RNase H1knockouts confer a temperature-sensitive phenotype,[7] and inS. cerevisiae, they produce defects in stress response.[23]
In prokaryotes, RNase H2 is enzymatically active as a monomeric protein. In eukaryotes, it is an obligate heterotrimer composed of a catalytic subunit A and structural subunits B and C. While the A subunit is closely homologous to the prokaryotic RNase H2, the B and C subunits have no apparent homologs in prokaryotes and are poorly conserved at thesequence level even among eukaryotes.[26][27] The B subunit mediatesprotein-protein interactions between the H2 complex andPCNA, which localizes H2 toreplication foci.[28]
Both prokaryotic and eukaryotic H2 enzymes can cleave single ribonucleotides in a strand.[2] however, they have slightly different cleavage patterns and substrate preferences: prokaryotic enzymes have lowerprocessivity and hydrolyze successive ribonucleotides more efficiently than ribonucleotides with a5' deoxyribonucleotide, while eukaryotic enzymes are more processive and hydrolyze both types of substrate with similar efficiency.[2][27] The substrate specificity of RNase H2 gives it a role inribonucleotide excision repair, removing misincorporated ribonucleotides from DNA, in addition toR-loop processing.[29][30][28] Although both H1 and H2 are present in the mammaliancell nucleus, H2 is the dominant source of RNase H activity there and is important for maintaining genome stability.[28]
Some prokaryotes possess an additional H2-type gene designated RNase HIII in the Roman-numeral nomenclature used for the prokaryotic genes. HIII proteins are more closely related to the H2 group bysequence identity and structural similarity, but have substrate preferences that more closely resemble H1.[7][31] Unlike HI and HII, which are both widely distributed among prokaryotes, HIII is found in only a few organisms with a scattered taxonomic distribution; it is somewhat more common inarchaea and is rarely or never found in the same prokaryotic genome as HI.[32]
Theactive site of nearly all RNases H contains four negatively charged amino acid residues, known as the DEDD motif; often ahistidine e.g. in HIV-1, human or E. coli is also present.[2][7]
The charged residues bind two metal ions that are required for catalysis; under physiological conditions these aremagnesium ions, butmanganese also usually supports enzymatic activity,[2][7] whilecalcium or high concentration of Mg2+ inhibits activity.[11][33][34]
Based on experimental evidence and computer simulations the enzyme activates a water molecule bound to one of the metal ions with the conserved histidine.[33][35] Thetransition state is associative in nature[17] and forms an intermediate with protonated phosphate and deprotonated alkoxide leaving group.[35] The leaving group is protonated via the glutamate which has an elevatedpKa and is likely to be protonated. The mechanism is similar toRNase T and the RuvC subunit in theCas9 enzyme which both also use a histidine and a two-metal ion mechanism.
The mechanism of the release of the cleaved product is still unresolved. Experimental evidence from time-resolved crystallography and similar nucleases points to a role of a third ion in the reaction recruited to the active site.[36][37]
RNASEH2A, the catalytic subunit of the trimeric H2 complex
RNASEH2B, a structural subunit of the trimeric H2 complex
RNASEH2C, a structural subunit of the trimeric H2 complex
In addition, genetic material ofretroviral origin appears frequently in the genome, reflecting integration of the genomes ofhuman endogenous retroviruses. Such integration events result in the presence of genes encoding retroviralreverse transcriptase, which includes an RNase H domain. An example isERVK6.[38]Long terminal repeat (LTR) and non-long terminal repeat (non-LTR)retrotransposons are also common in the genome and often include their own RNase H domains, with a complex evolutionary history.[39][40][41]
The structure of the trimeric human H2 complex, with the catalytic A subunit in blue, the structural B subunit in brown, and the structural C subunit in pink. Although the B and C subunits do not interact with the active site, they are required for activity. The catalytic residues in theactive site are shown in magenta. Positions shown in yellow are those with known AGS mutations. The most common AGS mutation -alanine tothreonine at position 177 of subunit B - is shown as a green sphere. Many of these mutations do not disrupt catalytic activityin vitro, but do destabilize the complex or interfere withprotein-protein interactions with other proteins in the cell.[42]
Mutations in any of the three RNase H2 subunits are well-established as causes of araregenetic disorder known asAicardi–Goutières syndrome (AGS),[3] which manifests asneurological anddermatological symptoms at an early age.[43] The symptoms of AGS closely resemble those of congenital viral infection and are associated with inappropriate upregulation oftype I interferon. AGS can also be caused by mutations in other genes:TREX1,SAMHD1,ADAR, andMDA5/IFIH1, all of which are involved in nucleic acid processing.[44] Characterization of mutational distribution in an AGS patient population found 5% of all AGS mutations in RNASEH2A, 36% in 2B, and 12% in 2C.[45] Mutations in 2B have been associated with somewhat milder neurological impairment[46] and with an absence of interferon-induced gene upregulation that can be detected in patients with other AGS-associated genotypes.[44]
The crystal structure of the HIV reverse transcriptase heterodimer (yellow and green), with the RNase H domain shown in blue (active site in magenta spheres). The orange nucleic acid strand is RNA, the red strand is DNA.[47]
Retroviral RT proteins fromHIV-1 andmurine leukemia virus are the best-studied members of the family.[50][51] Retroviral RT is responsible for converting the virus' single-stranded RNA genome into double-stranded DNA. This process requires three steps: first,RNA-dependent DNA polymerase activity producesminus-strand DNA from the plus-strand RNA template, generating an RNA:DNA hybrid intermediate; second, the RNA strand is destroyed; and third,DNA-dependent DNA polymerase activity synthesizes plus-strand DNA, generating double-stranded DNA as the final product. The second step of this process is carried out by an RNase H domain located at theC-terminus of the RT protein.[5][6][52][53]
RNase H performs three types of cleaving actions: non-specific degradation of the plus-strand RNA genome, specific removal of the minus-strandtRNA primer, and removal of the plus-strand purine-rich polypurine tract (PPT) primer.[54] RNase H plays a role in the priming of the plus-strand, but not in the conventional method of synthesizing a new primer sequence. Rather RNase H creates a "primer" from the PPT that is resistant to RNase H cleavage. By removing all bases but the PPT, the PPT is used as a marker for the end of the U3 region of itslong terminal repeat.[53]
Because RNase H activity is required for viral proliferation, this domain has been considered adrug target for the development ofantiretroviral drugs used in the treatment ofHIV/AIDS and other conditions caused by retroviruses.Inhibitors of retroviral RNase H of several differentchemotypes have been identified, many of which have amechanism of action based onchelation of the active-site cations.[55]Reverse-transcriptase inhibitors that specifically inhibit the polymerase function of RT are in widespread clinical use, but not inhibitors of the RNase H function; it is the only enzymatic function encoded by HIV that is not yet targeted by drugs in clinical use.[52][56]
RNases H are widely distributed and occur in alldomains of life. The family belongs to a larger superfamily ofnuclease enzymes[8][9] and is considered to be evolutionarily ancient.[57] In prokaryotic genomes, multiple RNase H genes are often present, but there is little correlation between occurrence of HI, HII, and HIII genes and overallphylogenetic relationships, suggesting thathorizontal gene transfer may have played a role in establishing the distribution of these enzymes. RNase HI and HIII rarely or never appear in the same prokaryotic genome. When an organism's genome contains more than one RNase H gene, they sometimes have significant differences in activity level. These observations have been suggested to reflect an evolutionary pattern that minimizes functional redundancy among RNase H genes.[7][32] RNase HIII, which is unique to prokaryotes, has a scattered taxonomic distribution and is found in bothbacteria andarchaea;[32] it is believed to have diverged from HII fairly early.[58]
The evolutionary trajectory of RNase H2 in eukaryotes, especially the mechanism by which eukaryotic homologs became obligate heterotrimers, is unclear; the B and C subunits have no apparent homologs in prokaryotes.[2][27]
Because RNase H specifically degrades only the RNA in double-stranded RNA:DNA hybrids, it is commonly used as alaboratory reagent inmolecular biology.Purified preparations ofE. coli RNase HI and HII are commercially available. RNase HI is often used to destroy the RNA template after first-strandcomplementary DNA (cDNA) synthesis byreverse transcription. It can also be used to cleave specific RNA sequences in the presence of short complementary segments of DNA.[59] Highly sensitive techniques such assurface plasmon resonance can be used for detection.[60][61] RNase HII can be used to degrade the RNA primer component of anOkazaki fragment or to introduce single-stranded nicks at positions containing a ribonucleotide.[59] A variant ofhot start PCR, known asRNase H-dependent PCR or rhPCR, has been described using a thermostable RNase HII from thehyperthermophilicarchaeonPyrococcus abyssi.[62] Of note, theribonuclease inhibitor protein commonly used as a reagent is not effective at inhibiting the activity of either HI or HII.[59]
Ribonucleases H were first discovered in the laboratory ofPeter Hausen when researchers found RNA:DNA hybridendonuclease activity incalfthymus in 1969 and gave it the name "ribonucleaseH" to designate itshybrid specificity.[26][63][64] RNase H activity was subsequently discovered inE. coli[65] and in a sample ofoncoviruses withRNA genomes during early studies of viralreverse transcription.[66][67] It later became clear that calf thymus extract contained more than one protein with RNase H activity[68] and thatE. coli contained two RNase H genes.[69][70] Originally, the enzyme now known as RNase H2 in eukaryotes was designated H1 and vice versa, but the names of the eukaryotic enzymes were switched to match those inE. coli to facilitate comparative analysis, yielding the modern nomenclature in which the prokaryotic enzymes are designated with Roman numerals and the eukaryotic enzymes with Arabic numerals.[2][26][31][71] The prokaryotic RNase HIII, reported in 1999, was the last RNase H subtype to be identified.[31]
Characterizing eukaryotic RNase H2 was historically a challenge, in part due to its low abundance.[2] Careful efforts atpurification of the enzyme suggested that, unlike theE. coli RNase H2, the eukaryotic enzyme had multiple subunits.[72] TheS. cerevisiae homolog of theE. coli protein (that is, the H2A subunit) was easily identifiable bybioinformatics when the yeastgenome was sequenced,[73] but the corresponding protein was found not to have enzymatic activity in isolation.[2][23] Eventually, the yeast B and C subunits were isolated by co-purification and found to be required for enzymatic activity.[74] However, the yeast B and C subunits have very lowsequence identity to their homologs in other organisms, and the corresponding human proteins were conclusively identified only after mutations in all three were found to causeAicardi–Goutières syndrome.[2][3]
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